1. Field of the Invention
The present invention generally relates to the field of methods for generating covalently interstrand bonding between nucleotides. More particularly, the present invention relates to the field of improved methods of directly covalent bond formation between homologous nucleotide sequences.
2. Description of the Prior Art
The following references are pertinent to this invention:
1. Falletta et.al., "Phase 1 Evaluation of Diaziquone in Childhood Cancer", Investigational New Drugs 8: 167-170 (1990). PA0 2. Hartley et.al., "DNA Cross-linking and Sequence Selectivity of Aziridinylbenzoquinones", Biochemistry 30: 11719-11724 (1991). PA0 3. Hampson et.al., "Chemical Crosslinking Subtraction; A New Method for the Generation of subtractive hybridization probes", Nucleic Acids Res. 20: 2899 (1992). PA0 4. Kimler et.al., "Combination of Aziridinylbenzoquinone and Cis-platinum with Radiation Therapy in the 9L Rat Brain Tumor Model", International Journal of Radiation Oncology, Biology, Physics 26: 445-450 (1993). PA0 5. Lehninger et.al., "Principles of Biochemistry, 2nd Edition", Worth Press, pp342-343 (1993). PA0 6. Lisitsyn et.al., "Cloning the Differences Between Two Complex Genomes", Science 259: 946-951 (1993). PA0 7. Sambrook et.al., "Molecular Cloning, 2nd Edition", Cold Spring Harbor Laboratory Press, p10.45 (1989). PA0 8. Solomons et.al., "Organic Chemistry, 6th Edition", John Wiley & Sons Press, pp 693, 803-804 (1996). PA0 9. Tan et.al., "Phase 1 Study of Azinridinylbenzoquinone in Children with Cancer", Cancer Research 44: 831-835 (1984). PA0 10. Wicland et.al., "A Method for Difference Cloning; Gene Amplification Following Subtractive Hybridization", Proc. Natl. Acad. Sci. USA 87: 2720-2724 (1990). PA0 11. Ueli et.al., "A Simple and Very Efficient Method for Generating cDNA Libraries", Gene, 25: pp263-269 (1983). PA0 12. U.S. Pat. No. 5,589,339 issued to Hampson. PA0 13. U.S. Pat. No. 5,591,575 issued to Hampson.
The ability to form covalent bonding between two nucleotide sequences has permitted a complete subtraction of common sequences during subtractive hybridization as well as a fully stable probing activity during in-situ-hybridization and antisense therapy. Because the covalent bonding is one of the strongest and most heat-stable interactions between molecules, the covalent bonding between two nucleotide sequences can sustain some harsh procedures, such as denature, salting and enzyme digestion. Based on such property, some methods have been developed either to perform chemotherapy or to isolate specific nucleotide sequences with external cross-linking chemicals by which two nucleotide strands were indirectly bonded. One of the most commonly used cross-linking chemicals to accomplish such sequence selectivity is aziridinylbenzoquinone (AZQ)-class agent (Hartley et.al., Biochemistry 30: 11719-11724 (1991)), involving the cross-linking of guanine and cytosine.
AZQ-class agents have been used in the chemotherapy of some cancers, such as brain tumor in rats (Kimler et.al., International Journal of Radiation Oncology, Biology, Physics 26: 445-450 (1993)) and phase 1 childhood cancer in human (Falletta et.al., Investigational New Drugs 8: 167-170 (1990); Tan et.al., Cancer Research 44: 831-835 (1984)). However, although AZQ successfully raises the bonding stability of double-stranded genome and reduces the replication of cancer cells, the non-specific cross-linking feature of AZQ also causes significant toxicity to the normal cells. Since the AZQ lacks sequence-specific targeting capability in vivo, some in vitro methods have been designed to detect and isolate specific nucleotide sequences with AZQ which cross-links common sequences of two compared nucleotide libraries.
Prior art attempts at simplifying subtraction with covalent affinity, such as U.S. Pat. No. 5,589,339 and U.S. Pat. No. 5,591,575 to Hampson, also uses an AZQ interstrand cross-linking agent to covalently subtract common sequences from a tester library. In brief, this method relies upon the generation of single-stranded tester and driver libraries which contain all sense or all antisense sequences. After the tester is hybridized with the driver, resulting in hybrid duplex formation if a sequence is common to both libraries, the AZQ is added to generate externally covalent bonds between the hybrid duplexes. Because the AZQ cross-links all double-stranded sequences, this kind of covalent-bonding nature greatly increases the completion of homologue subtraction after hybridization. However, in this method, both of the initial tester and driver must be all single strands due to the interstrand cross-linking nature of the AZQ-class agents, resulting in no use of genomic DNA samples, no detection of limited initial materials and no specific-primer amplification of final results. These disadvantages cause more restrictions of sample selection, less stability of sample storage and less sensitivity of final result detection in comparison with traditional methods. Also, the determination of final desired sequences is completed by a non-specific random-primer extension reaction which lowers the specificity of final results.
In summary, it is desirable to have a fast, specific and direct covalent bonding method for subtracting common sequences in a subtractive hybridization procedure as well as for increasing probing specificity in a gene targeting system, of which the results may contribute to developing a screening method for new genes, a diagnosis for inherent problems, or a therapy for diseases.